U.S. patent application number 14/353815 was filed with the patent office on 2014-10-09 for methyltransferase inhibitors for treating cancer.
The applicant listed for this patent is MEMORIAL SLOAN-KETTERING CANCER CENTER. Invention is credited to Glorymar del Valle Ibanzen Sanchez, Minkui Luo, Weihong Zheng.
Application Number | 20140303106 14/353815 |
Document ID | / |
Family ID | 47178343 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140303106 |
Kind Code |
A1 |
Zheng; Weihong ; et
al. |
October 9, 2014 |
METHYLTRANSFERASE INHIBITORS FOR TREATING CANCER
Abstract
Compounds having methyltransferase inhibitory activity are
disclosed. The compounds have the structure ##STR00001## and are
useful in the treatment of cancer and similar diseases associated
with inappropriate methyltransferase activity.
Inventors: |
Zheng; Weihong; (New York,
NY) ; Luo; Minkui; (New York, NY) ; Ibanzen
Sanchez; Glorymar del Valle; (Corona, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEMORIAL SLOAN-KETTERING CANCER CENTER |
New York |
NY |
US |
|
|
Family ID: |
47178343 |
Appl. No.: |
14/353815 |
Filed: |
October 26, 2012 |
PCT Filed: |
October 26, 2012 |
PCT NO: |
PCT/US2012/062157 |
371 Date: |
April 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61551976 |
Oct 27, 2011 |
|
|
|
61552236 |
Oct 27, 2011 |
|
|
|
61624636 |
Apr 16, 2012 |
|
|
|
Current U.S.
Class: |
514/46 ; 435/184;
514/49; 536/27.2; 536/27.3 |
Current CPC
Class: |
C07D 473/34 20130101;
C07H 19/16 20130101; A61P 35/00 20180101; C07D 487/04 20130101;
C07H 19/14 20130101 |
Class at
Publication: |
514/46 ;
536/27.3; 536/27.2; 514/49; 435/184 |
International
Class: |
C07H 19/14 20060101
C07H019/14; C07H 19/16 20060101 C07H019/16 |
Claims
1. A compound of formula I or II ##STR00024## wherein: X.sup.1 is N
or CH; Q is NH or O; A is chosen from direct bond,
(C.sub.1-C.sub.20)hydrocarbon, (C.sub.1-C.sub.20)oxaalkyl and
(C.sub.1-C.sub.20)azaalkyl; R.sup.1 is chosen from hydrogen,
--C(.dbd.NH)NH.sub.2, --C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon,
fluoro(C.sub.1-C.sub.6)hydrocarbon, and --CH(NH.sub.2)COOH, with
the provisos that, (1) when A is a direct bond, R.sup.1 cannot be
H; (2) when QR.sup.3 is OH, R.sup.1 cannot be
fluoro(C.sub.2-C.sub.6)hydrocarbon; R.sup.2 is chosen from
hydrogen, --C(.dbd.NH)NH.sub.2,
--C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon and --CH(NH.sub.2)COOH;
R.sup.3 is chosen from H and (C.sub.1-C.sub.20) hydrocarbon; and n
is 1 or 2.
2. A compound according to claim 1 wherein R.sup.3 is chosen from
H, methyl and ethyl.
3. A compound according to claim 1 wherein n is 2.
4. A compound according to claim 3 wherein QR.sup.3 is OH.
5. A compound according to claim 1 of formula Ia or IIa
##STR00025## wherein: X.sup.1 is N or CH; A is chosen from direct
bond, (C.sub.1-C.sub.20)hydrocarbon, (C.sub.1-C.sub.20)oxaalkyl and
(C.sub.1-C.sub.20)azaalkyl; R.sup.1 is chosen from hydrogen,
--C(.dbd.NH)NH.sub.2, --C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon,
CF.sub.3 and --CH(NH.sub.2)COOH, with the proviso that, when A is a
direct bond, R.sup.1 cannot be H; R.sup.2 is chosen from hydrogen,
--C(.dbd.NH)NH.sub.2, --C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon
and --CH(NH.sub.2)COOH.
6. A compound according to claim 1 of formula I or Ia wherein
R.sup.1-A is chosen from (C.sub.1-C.sub.6)alkyl, benzyl and
(C.sub.3-C.sub.6)oxaalkyl.
7. A compound according to claim 1 of formula I or Ia wherein
R.sup.1-A is chosen from amino(C.sub.1-C.sub.6)alkyl,
benzylamino(C.sub.1-C.sub.6)alkyl and
guanidino(C.sub.1-C.sub.6)alkyl.
8. A compound according to claim 1 of formula I or Ia wherein
R.sup.1-A is chosen from HOOC(NH.sub.2)CH-azaalkyl and
NH.sub.2(NH.dbd.)C-azaalkyl.
9. A compound according to claim 1 of formula II or IIa wherein
R.sup.2-A is chosen from hydrogen, (C.sub.1-C.sub.6)alkyl, benzyl
and --C(.dbd.NH)NH.sub.2.
10. A compound according to claim 1 wherein X.sup.1 is N.
11. A compound according to claim 1 wherein X.sup.1 is CH.
12. A method for inhibiting the activity of a methyltransferase
enzyme comprising bringing said methyltransferase enzyme into
contact with a compound according to claim 1.
13. A method for selectively inhibiting the activity of a first
methyltransferase enzyme in the presence of a second
methyltransferase enzyme comprising bringing both of said
methyltransferase enzymes into contact with a compound according to
claim 1.
14. A method of treating cancer in a patient suffering from cancer
comprising administering to said patient a therapeutically
effective amount of a compound according to claim 1.
15. A method according to claim 14 wherein said cancer is breast
cancer or prostate cancer.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
applications 61/551,976; 61/552,236; and 61/624,636; filed Oct. 27,
2011; Oct. 27, 2011; and Apr. 16, 2012, respectively. The entire
contents of all three are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to chemical compounds having
methyltransferase inhibitory activity and their use in the
treatment of diseases and conditions associated with inappropriate
methyltransferase activity.
BACKGROUND OF THE INVENTION
[0003] Epigenetics is inheritable information not encoded in DNA
manifested through control of gene expression, thereby controlling
a range of cellular activity, including determining cell fate, stem
cell fate and regulating proliferation. Epigenetic control over
gene expression is accomplished in at least four ways: (1) covalent
histone modification, (2) covalent DNA modification, (3) histone
variation, and (4) nucleosome structure and DNA/histone contact
points. Epigenetic control through one mechanism can influence the
other suggesting a combinatorial regulation, as evidenced by the
methylation of histones being implicated in the modulation of DNA
methylation.
[0004] Covalent histone modifications, a key mechanism involved in
epigenetic control, include: (1) lysine acetylation, (2) lysine and
arginine methylation, (3) serine and threonine phosphorylation, (4)
ADP-ribosylation, (5) ubiquitination, and (6) SUMOylation. Specific
enzymatic activities are associated with these modifications and in
the case of histone methylation, methyltransferases catalyze the
transfer of a methyl group from cofactor S-adenosylmethionine to a
lysine or arginine, producing S-adenosylhomocysteine as a
by-product. Methyltransferases can also modify residues in other
cellular proteins, e.g. the tumor suppressor p53.
[0005] Histone methyltransferases fall into subgroups that include
arginine methyltransferases, SET-domain containing
methyltransferases SU(VAR)3-9, E(Z) and TRX, and DOT-like
methyltransferase hDOT1L. Families of SET-domain containing
methyltransferases have been identified and include SUV39, SET1,
SET2 and RIZ.
[0006] The disruption of the normal functions of methyltransferases
has been implicated in human diseases. Members of different classes
of methyltransferases are implicated in cancer and representative
examples for the subgroups and subclasses are provided: (1) hDOT1L,
a member of the DOT-like methyltransferases, is linked to
leukemogenesis [Nature Cell Biology, 8:1017-1028 (2006); Cell,
121:167-178 (2005); Cell, 112:771-723 (2003)]. (2) EZH2, a SET1
methyltransferase, is up-regulated in tumor cell lines and has been
linked to breast, gastric and prostate cancers [British Journal of
Cancer, 90:761-769 (2004)]. (3) SUV39-1/2, SUV39
methyltransferases, have been linked to signaling pathways
regulating cancer cell growth and differentiation [Genetica,
117(2-3):149-58 (2003)]. (4) NSD1, a SET2 subclass
methyltransferase, has been linked to acute myeloid leukemia and
Sotos syndrome, a predisposition to cancer [Molecular Cell Biology,
24(12):5184-96 (2004)]. (5) EVI1, a RIZ methyltransferase, is
overexpressed in solid tumors and leukemia [Proceeding of the
National Academy of Sciences, 93:1642-1647 (1996)]. (6) Related
enzymes, namely SMYD2, are lysine methyltransferases that modify
the tumor suppressor protein, p53 and through this activity, may
function as an oncogene that interferes with p53's protective
functions [Nature, 444(7119):629-632 (2006)]. (7) SMYD3, a
SET-domain containing lysine methyltransferase, is involved in
cancer cell proliferation [Nature Cell Biology, 6(8):731-740
(2004)]. (8) CARM1, an arginine methlytransferase, is linked to
prostate cancer [Prostate, 66(12):1292-301 (2006)].
[0007] Inappropriate methyltransferase activities thus represent
attractive targets for therapeutic intervention by small molecule
inhibitors. In fact, inhibitors of SUV(AR) histone
methyltransferase [Nature Chemical Biology, 1:143-145 (2005)] and
protein arginine methyltransferase [Journal of Biological
Chemistry, 279:23892-23899 (2004)] have been described. The present
invention relates to novel synthetic compounds effective as
inhibitors of inappropriate histone methyltransferase activities
that would be useful in treating human diseases, such as
cancer.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention relates to compounds of general
formulae I and II, which are potent and selective inhibitors of
lysine and arginine methyltransferases:
##STR00002##
wherein: [0009] X.sup.1 is N or CH; [0010] Q is NH or O; [0011] A
is chosen from direct bond, (C.sub.1-C.sub.20)hydrocarbon,
(C.sub.1-C.sub.20)oxaalkyl and (C.sub.1-C.sub.20)azaalkyl; [0012]
R.sup.1 is chosen from hydrogen, --C(.dbd.NH)NH.sub.2,
--C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon,
fluoro(C.sub.1-C.sub.6)hydrocarbon, and --CH(NH.sub.2)COOH, with
the provisos that, [0013] (1) when A is a direct bond, R.sup.1
cannot be H; [0014] (2) when QR.sup.3 is OH, R.sup.1 cannot be
fluoro(C.sub.2-C.sub.6)hydrocarbon; [0015] R.sup.2 is chosen from
hydrogen, --C(.dbd.NH)NH.sub.2,
--C(.dbd.NH)NH(C.sub.1-C.sub.10)hydrocarbon and --CH(NH.sub.2)COOH;
[0016] R.sup.3 is chosen from H and (C.sub.1-C.sub.20) hydrocarbon;
and [0017] n is 1 or 2.
[0018] In these compounds, A is a bivalent moiety and R.sup.1 or
R.sup.2 is a substituent on A. The members of these genera are
effective as inhibitors of methyltransferase activities and
therefore, are useful for the inhibition, prevention and
suppression of various pathologies associated with such activities,
such as, for example, cancer cell and cancer stem cell fate
differentiation, and cancer cell proliferation and cell cycle
regulation. The compounds are also useful research tools for
studying protein methyl transferase biology.
[0019] In another aspect, the invention relates to pharmaceutical
compositions comprising a therapeutically effective amount of at
least one compound of general formula I or II and a
pharmaceutically acceptable carrier.
[0020] In another aspect, the invention relates to a method for
treating cancer comprising administering to a subject suffering
from a cancer a therapeutically effective amount of a compound of
formula I or II.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Throughout this specification the substituents are defined
when introduced and retain their definitions.
[0022] In one aspect, the invention relates to compounds having
general formula I:
##STR00003##
[0023] In some embodiments of I, R.sup.3 is chosen from H, methyl
and ethyl. In some embodiments n is 2. In some embodiments QR.sup.3
is OH. In some embodiments n is 1 and QR.sup.3 is OH; these fall
into a genus of formula Ia:
##STR00004##
[0024] In some embodiments, R.sup.1-A is chosen from
(C.sub.1-C.sub.6)alkyl, benzyl and (C.sub.3-C.sub.6)oxaalkyl. In
these embodiments, R.sup.1 is conceptually H and A is, for example,
--(CH.sub.2CH.sub.2CH.sub.2)--; or R.sup.1 is conceptually H and A
is
##STR00005##
or R.sup.1 is H and A is --(CH.sub.2OCH.sub.2CH.sub.2CH.sub.2)--.
In other embodiments, R.sup.1-A is chosen from
amino(C.sub.1-C.sub.6)alkyl, benzylamino(C.sub.1-C.sub.6)alkyl and
guanidino(C.sub.1-C.sub.6)alkyl. In this latter compound, R.sup.1
is --C(.dbd.NH)NH.sub.2 and A is considered an azaalkyl, for
example, --NHCH.sub.2CH.sub.2--.
[0025] In some embodiments R.sup.1-A may be chosen from
HOOC(NH.sub.2)CH-azaalkyl and NH.sub.2(NH.dbd.)C-azaalkyl.
[0026] In another aspect the invention relates to compounds having
general formula II
##STR00006##
[0027] In some embodiments of II, R.sup.3 is chosen from H, methyl
and ethyl. In some embodiments n is 2. In some embodiments QR.sup.3
is OH. In some embodiments n is 1 and QR.sup.3 is OH; these fall
into a genus of formula IIa:
##STR00007##
[0028] In some embodiments R.sup.2-A is chosen from hydrogen,
(C.sub.1-C.sub.6)alkyl, benzyl and --C(.dbd.NH)NH.sub.2.
When A is azaalkyl, the number of carbons between nitrogens is
preferably two or three. Thus, R.sup.1-A and R.sup.2-A may be, for
example, aminoethyl, benzylaminoethyl, guanidinoethyl, and
(C.sub.1-C.sub.6)alkyaminoethyl.
[0029] In all of the foregoing embodiments, X.sup.1 may be CH, i.e.
the heterocycle is 7-deazapurine (also known as
7H-pyrrolo[2,3-d]pyrimidine) or X.sup.1 may be N, i.e. the
heterocycle is purine.
[0030] For convenience and clarity certain terms employed in the
specification, examples and claims are described herein.
[0031] Unless otherwise specified, alkyl (or alkylene) is intended
to include linear, branched, or cyclic hydrocarbon structures and
combinations thereof. A combination would be, for example,
cyclopropylmethyl. Lower alkyl refers to alkyl groups of from 1 to
6 carbon atoms. Examples of lower alkyl groups include methyl,
ethyl, propyl, isopropyl, butyl, s- and t-butyl and the like.
Preferred alkyl groups are those of C.sub.10 or below. Cycloalkyl
is a subset of alkyl and includes cyclic hydrocarbon groups of from
3 to 8 carbon atoms. Examples of cycloalkyl groups include
c-propyl, c-butyl, c-pentyl, norbornyl and the like.
[0032] C.sub.1 to C.sub.20 hydrocarbon includes alkyl, cycloalkyl,
polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof.
Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl,
camphoryl and naphthylethyl. Hydrocarbon refers to any substituent
comprised of hydrogen and carbon as the only elemental
constituents.
[0033] Unless otherwise specified, the term "carbocycle" is
intended to include ring systems in which the ring atoms are all
carbon but of any oxidation state. Thus (C.sub.3-C.sub.10)
carbocycle refers to both non-aromatic and aromatic systems,
including such systems as cyclopropane, benzene and cyclohexene;
(C.sub.8-C.sub.12) carbopolycycle refers to such systems as
norbornane, decalin, indane and naphthalene. Carbocycle, if not
otherwise limited, refers to monocycles, bicycles and
polycycles.
[0034] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon
atoms of a straight, branched or cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to
groups containing one to four carbons. For the purpose of this
application, alkoxy and lower alkoxy include methylenedioxy and
ethylenedioxy.
[0035] Oxaalkyl refers to alkyl residues in which one or more
carbons (and their associated hydrogens) have been replaced by
oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the
like. The term oxaalkyl is intended as it is understood in the art
[see Naming and Indexing of Chemical Substances for Chemical
Abstracts, published by the American Chemical Society, 196, but
without the restriction of 127(a)], i.e. it refers to compounds in
which the oxygen is bonded via a single bond to its adjacent atoms
(forming ether bonds); it does not refer to doubly bonded oxygen,
as would be found in carbonyl groups. Similarly, thiaalkyl and
azaalkyl refer to alkyl residues in which one or more carbons has
been replaced by sulfur or nitrogen, respectively. Examples of
azaalkyl include ethylaminoethyl and aminohexyl.
[0036] Substituents R.sup.n are generally defined when introduced
and retain that definition throughout the specification and in all
independent claims.
[0037] As used herein, and as would be understood by the person of
skill in the art, the recitation of "a compound"--unless expressly
further limited--is intended to include salts of that compound.
Thus, for example, the recitation "a compound of formula I" as
depicted above, which depicts a substituent COOH, would include
salts in which the substituent is COO.sup.-M.sup.+, wherein M is
any counterion. Similarly, formula I as depicted above depicts a
substituent NH.sub.2, and therefore would also include salts in
which the substituent is NH.sub.3.sup.+X.sup.-, wherein X is any
counterion. The compounds may commonly exist as zwitterions, which
are effectively internal salts. In a particular embodiment, the
term "compound of formula I" refers to the compound or a
pharmaceutically acceptable salt thereof. As used herein, and as
would be understood by the person of skill in the art, the
recitation of "a compound"--unless expressly further limited--is
intended to include salts of that compound. In a particular
embodiment, the term "compound of formula I" refers to the compound
or a pharmaceutically acceptable salt thereof.
[0038] The term "pharmaceutically acceptable salt" refers to salts
whose counter ion derives from pharmaceutically acceptable
non-toxic acids and bases. Suitable pharmaceutically acceptable
acids for salts of the compounds of the present invention include,
for example, acetic, adipic, alginic, ascorbic, aspartic,
benzenesulfonic (besylate), benzoic, boric, butyric, camphoric,
camphorsulfonic, carbonic, citric, ethanedisulfonic,
ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric,
glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric,
hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic,
laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic,
naphthylenesulfonic, nitric, oleic, pamoic, pantothenic,
phosphoric, pivalic, polygalacturonic, salicylic, stearic,
succinic, sulfuric, tannic, tartaric acid, teoclatic,
p-toluenesulfonic, and the like. Suitable pharmaceutically
acceptable base addition salts for the compounds of the present
invention include, but are not limited to, metallic salts made from
aluminum, calcium, lithium, magnesium, potassium, sodium and zinc
or organic salts made from lysine, arginine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium cations and carboxylate, sulfonate
and phosphonate anions attached to alkyl having from 1 to 20 carbon
atoms.
[0039] It will be recognized that the compounds of this invention
can exist in radiolabeled form, i.e., the compounds may contain one
or more atoms containing an atomic mass or mass number different
from the atomic mass or mass number usually found in nature.
Alternatively, a plurality of molecules of a single structure may
include at least one atom that occurs in an isotopic ratio that is
different from the isotopic ratio found in nature. Radioisotopes of
hydrogen, carbon, phosphorous, fluorine, chlorine and iodine
include .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N,
.sup.35S, .sup.18F, .sup.36Cl, .sup.125I, .sup.124I and .sup.131I
respectively. Compounds that contain those radioisotopes and/or
other radioisotopes of other atoms are within the scope of this
invention. Tritiated, i.e. .sup.3H, and carbon-14, i.e., .sup.14C,
radioisotopes are particularly preferred for their ease in
preparation and detectability. Compounds that contain isotopes
.sup.11C, .sup.13N, .sup.15O, .sup.124I and .sup.18F are well
suited for positron emission tomography. Radiolabeled compounds of
formulae I and II of this invention and prodrugs thereof can
generally be prepared by methods well known to those skilled in the
art. Conveniently, such radiolabeled compounds can be prepared by
carrying out the procedures disclosed in the Examples and Schemes
by substituting a readily available radiolabeled reagent for a
non-radiolabeled reagent.
[0040] Although this invention is susceptible to embodiment in many
different forms, preferred embodiments of the invention are shown.
It should be understood, however, that the present disclosure is to
be considered as an exemplification of the principles of this
invention and is not intended to limit the invention to the
embodiments illustrated. It may be found upon examination that
certain members of the claimed genus are not patentable to the
inventors in this application. In this event, subsequent exclusions
of species from the compass of applicants' claims are to be
considered artifacts of patent prosecution and not reflective of
the inventors' concept or description of their invention; the
invention encompasses all of the members of the genera I and II
that are not already in the possession of the public.
[0041] While it may be possible for the compounds of formula I or
II to be administered as the raw chemical, it is preferable to
present them as a pharmaceutical composition. According to a
further aspect, the present invention provides a pharmaceutical
composition comprising a compound of formula I or II or a
pharmaceutically acceptable salt or solvate thereof, together with
one or more pharmaceutically carriers thereof and optionally one or
more other therapeutic ingredients. The carrier(s) must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof. The compositions may be formulated for oral, topical or
parenteral administration. For example, they may be given
intravenously, intraarterially, subcutaneously, and directly into
the CNS--either intrathecally or intracerebroventricularly.
[0042] Formulations include those suitable for oral, parenteral
(including subcutaneous, intradermal, intramuscular, intravenous
and intraarticular), rectal and topical (including dermal, buccal,
sublingual and intraocular) administration. The compounds are
preferably administered orally or by injection (intravenous or
subcutaneous). The precise amount of compound administered to a
patient will be the responsibility of the attendant physician.
However, the dose employed will depend on a number of factors,
including the age and sex of the patient, the precise disorder
being treated, and its severity. Also, the route of administration
may vary depending on the condition and its severity. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. In general, the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both and then,
if necessary, shaping the product into the desired formulation.
[0043] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The
active ingredient may also be presented as a bolus, electuary or
paste.
[0044] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example those suitable for
oral administration may include flavoring agents.
[0045] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" are used interchangeably herein. These terms
refers to an approach for obtaining beneficial or desired results
including but not limited to therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological systems associated
with the underlying disorder such that an improvement is observed
in the patient, notwithstanding that the patient may still be
afflicted with the underlying disorder. For prophylactic benefit,
the compositions may be administered to a patient at risk of
developing a particular disease, or to a patient reporting one or
more of the physiological systems of a disease, even though a
diagnosis of this disease may not have been made.
[0046] Terminology related to "protecting", "deprotecting" and
"protected" functionalities occurs throughout this application.
Such terminology is well understood by persons of skill in the art
and is used in the context of processes that involve sequential
treatment with a series of reagents. In that context, a protecting
group refers to a group which is used to mask a functionality
during a process step in which it would otherwise react, but in
which reaction is undesirable. The protecting group prevents
reaction at that step, but may be subsequently removed to expose
the original functionality. The removal or "deprotection" occurs
after the completion of the reaction or reactions in which the
functionality would interfere. Thus, when a sequence of reagents is
specified, as it is in the processes of the invention, the person
of ordinary skill can readily envision those groups that would be
suitable as "protecting groups". Suitable groups for that purpose
are discussed in standard textbooks in the field of chemistry, such
as Protective Groups in Organic Synthesis by T. W. Greene [John
Wiley & Sons, New York, 1991], which is incorporated herein by
reference.
[0047] A comprehensive list of abbreviations utilized by organic
chemists appears in the first issue of each volume of the Journal
of Organic Chemistry. The list, which is typically presented in a
table entitled "Standard List of Abbreviations", is incorporated
herein by reference.
[0048] In general, the compounds of the present invention may be
prepared by the methods illustrated in the general reaction schemes
as, for example, described below, or by modifications thereof,
using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants that are in themselves known, but
are not mentioned here. The starting materials are either
commercially available, synthesized as described in the examples or
may be obtained by the methods well known to persons of skill in
the art.
##STR00008## ##STR00009##
##STR00010## ##STR00011##
##STR00012##
##STR00013##
##STR00014##
##STR00015##
##STR00016##
##STR00017##
[0049] In analagous fashion to that shown in Scheme 7, compound 205
was synthesized from intermediate 32:
##STR00018##
##STR00019##
##STR00020##
[0050] Deazapurines were synthesized as shown in Scheme 12:
##STR00021##
[0051] In the deprotection Step 1. for 115d, a by-product was
isolated in which the acetates were cleaved but the methyl ester
was not. These were separated, and in a subsequent Step 2, the CBZ
was cleaved and the allyl group was reduced to provide, in addition
to the fully deprotected and reduced products 116a-116d, the methyl
ester of the acid 116d, which is identified as 116e:
##STR00022##
[0052] Compounds in which n is 2 are synthesized as described in
Scheme 13:
##STR00023##
[0053] Synthesis of 4a, 4b, 4c. To a stirred suspension of sodium
hydride (60%, 86 mg, 2.1 mmol) in 5 mL THF at ambient temperature
was added dropwise the solution of the urethane 3 (160 mg, 0.36
mmol) in 20 mL THF. After the mixture was stirred for 1 h, the
corresponding halides (2.1 mmol) were added (methyl/ethyl/ally
iodide or benzyl bromide), followed by tetrabutylammonium iodide
(10 mg). The resultant mixture was stirred for 20 h. The reaction
was then quenched with saturated aqueous NH.sub.4Cl (20 mL) and
volatile components in the mixture was removed under reduced
pressure. This mixture was further extracted with ethyl acetate
(3.times.30 mL). The combined organic phase was washed with brine
and then dried with anhydrous Na.sub.2SO.sub.4. After removing
volatile components, the crude mixture was purified by silica gel
chromatography (hexane:EtOAc=3:1 then 2:1) to yield Compounds 4a,
4b, 4c as colorless oil.
[0054] 4a R=Me, 85% yield. [.alpha.].sub.D.sup.18.9+7.83 (c 1.30,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 74.degree. C.):
.delta. 1.25 (s, 3H), 1.38 (s, 3H), 1.60-1.64 (m, 1H), 1.78-1.82
(m, 1H), 2.17-2.21 (m, 1H), 2.30-2.31 (m, 1H), 2.75 (s, 1H), 3.27
(s, 3H), 3.96 (dd, 1H, J=10.4 Hz, 4.2 Hz), 4.22-4.23 (m, 1H), 4.54
(d, 1H, J=5.9 Hz), 4.56 (d, 1H, J=5.9 Hz), 4.85 (s, 1H), 4.99 (dt,
1H, J=10.2 Hz, 0.9 Hz), 5.05 (d, 1H, J=10.2 Hz), 5.09 (d, 2H, J=1.2
Hz) 5.63-5.72 (m, 1H), 7.28-7.31 (m, 1H), 7.33-7.36 (m, 4H);
.sup.13C-NMR (125 MHz, CDCl.sub.3, rotamers): .delta. 24.61, 24.96,
26.41, 26.47, 36.77, 37.23, 37.35, 37.64, 55.15, 55.30, 66.89,
67.24, 83.75, 83.82, 84.38, 84.48, 85.55, 85.60, 109.86, 110.00,
112.26, 112.35, 117.24, 117.40, 127.65, 127.80, 127.87, 128.31,
128.41, 134.57, 134.89, 136.92, 137.07, 165.42, 165.74; MS (ESI)
m/z: 428 [M+Na].sup.+; HRMS: calculated for
C.sub.22H.sub.31NO.sub.6Na ([M+Na].sup.+) 428.2049. found
428.2036.
[0055] 4b R=Et, 57% yield. [.alpha.].sub.D.sup.18.9+3.38 (c 0.87,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 74.degree. C.):
.delta. 1.11 (t, 3H, J=7.0 Hz), 1.26 (s, 3H), 1.38 (s, 3H),
1.63-1.67 (m, 1H), 1.89-1.91 (m, 1H), 2.23-2.27 (m, 1H), 2.35-2.40
(m, 1H), 3.18-3.23 (m, 2H), 3.27 (s, 3H), 3.96-3.98 (m, 1H), 4.01
(dd, 1H, J=10.7 Hz, 3.6 Hz), 4.54 (d, 1H, J=6.2 Hz), 4.55 (d, 1H,
J=5.9 Hz), 4.85 (s, 1H), 4.99 (dt, 1H, J=10.2 Hz, 1.0 Hz), 5.05
(dd, 1H, J=10.2 Hz, 1.7 Hz), 5.10 (s, 2H), 5.68-5.75 (m, 1H),
7.29-7.31 (m, 1H), 7.34-7.35 (m, 4H); .sup.13C-NMR (150 MHz,
DMSO-d.sub.6 rotamers): .delta. 14.35, 15.22, 24.64, 26.25, 36.50,
37.05, 37.59, 38.10, 54.49, 65.78, 66.08, 83.24, 83.32, 83.75,
84.81, 109.08, 109.21, 111.33, 117.08, 117.20, 127.25, 127.36,
127.70, 128.30, 128.40, 135.39, 135.58, 137.04, 137.25, 155.21,
155.39; MS (ESI) m/z: 442 [M+Na].sup.+, HRMS: calculated for
C.sub.23H.sub.33NO.sub.6Na ([M+Na].sup.+) 442.2206. found
442.2206.
[0056] 4c R=Bn, 86% yield. .sup.1H-NMR (600 MHz, DMSO-d.sub.6,
74.degree. C.): .delta. 1.23 (s, 3H), 1.36 (s, 3H), 1.61-1.66 (m,
1H), 1.86-1.89 (m, 1H), 2.22-2.26 (m, 1H), 2.28-2.31 (m, 1H), 3.22
(s, 3H), 3.94-3.96 (m, 2H), 4.33 (s, 1H), 4.36-4.38 (m, 2H), 4.48
(d, 1H, J=5.9 Hz), 4.54 (d, 1H, J=15.6 Hz), 4.83 (s, 1H), 4.90 (s,
1H), 4.92 (d, 1H, J=5.2 Hz), 5.14 (s, 2H), 5.53-5.60 (m, 1H),
7.23-7.24 (m, 1H), 7.29-7.34 (m, 9H); .sup.13C-NMR (150 MHz,
DMSO-d.sub.6 rotamers): .delta.24.65, 26.23, 36.45, 37.12, 37.46,
38.05, 54.51, 66.18, 66.60, 83.08, 83.21, 83.56, 84.75, 109.08,
111.27, 117.09, 126.96, 127.44, 127.56, 127.74, 128.23, 128.29,
135.16, 135.33, 136.80, 138.89, 155.30, 156.51; MS (ESI) m/z: 504
[M+Na].sup.+.
[0057] Synthesis of 5. To a stirred CH.sub.2Cl.sub.2 solution
containing the alkene 4 (0.29 mmol) was bubbled a stream of ozone
at -78.degree. C. until the blue color persisted over 5 min. After
the solution was flushed with argon and turned transparent,
triphenylphosphine (220 mg, 0.87 mmol) was added at -78.degree. C.
The dry ice bath was then removed and the reaction mixture was
allowed to warm up spontaneously at ambient temperature. The
resultant reaction mixture was stirred until the ozonide
intermediates disappeared (monitored by TLC, .about.20 h).
Evaporation of the solvent under reduced pressure gave the crude
aldehyde, which was purified by flash silica gel chromatography
(hexane:EtOAc=2:1 then 1:1) to yield the product as colorless oil.
Without further storage, the intermediated aldehyde (around 0.22
mmol) was dissolved in 15 mL ethanol and reacted with NaBH.sub.4
(10 mg, 0.29 mmol, added by batch) at 0.degree. C. under argon. The
reaction mixture was stirred at 0.degree. C. for 20 min and then
quenched with saturated aqueous NH.sub.4Cl solution (20 mL, added
dropwise). The resultant mixture was diluted with 20 mL ethyl
acetate. The organic phase was separated and the aqueous phase was
further extracted with ethyl acetate (3.times.30 mL). The combined
organic phase was washed with brine, dried with Na.sub.2SO.sub.4
and concentrated. The crude mixture was purified by a short column
chromatography (hexane:EtOAc=1:1 then 1:2) to give the
corresponding alcohols 5a, 5b and 5c.
[0058] 5b R=Et, 95% yield. [.alpha.].sub.D.sup.17.9+1.38 (c 1.17,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6 74.degree. C.):
.delta. 1.11 (t, 3H, J=7.0 Hz), 1.26 (s, 3H), 1.38 (s, 3H),
1.59-1.66 (m, 2H), 1.80-1.83 (m, 1H), 1.91-1.93 (m, 1H), 3.20-3.27
(m, 2H), 3.38 (s, 3H), 3.40 (q, 2H, J=6.0 Hz), 3.97 (br, 1H), 4.01
(dd, 1H, J=10.6 Hz, 3.7 Hz), 4.10-4.11 (m, 1H), 4.53 (d, 1H, J=6.0
Hz), 4.55 (d, 1H, J=6.0 Hz), 4.86 (s, 1H), 5.09 (s, 2H), 7.29-7.31
(m, 1H), 7.34-7.36 (m, 4H); .sup.13C-NMR (150 MHz, DMSO-d.sub.6
rotamers): .delta.4.92, 24.66, 26.25, 37.11, 37.75, 54.41, 57.79,
57.86, 65.69, 66.02, 83.43, 83.75, 84.81, 109.04, 109.14, 111.30,
127.23, 127.68, 128.26, 128.40, 137.27, 155.45; MS (ESI) m/z: 446
[M+Na].sup.+; HRMS: calculated for C.sub.22H.sub.33NO.sub.7Na
([M+Na].sup.+) 446.2155. found 446.2148.
[0059] Synthesis of 6. To the solution of the primary alcohol 5
(0.4 mmol) in 20 mL dry dichloromethane (DCM) was added triethyl
amine (82 .mu.L, 0.59 mmol) and then methanesulfonyl chloride (39
.mu.L, 0.5 mmol) at 0.degree. C. The resultant mixture was stirred
at 0.degree. C. for additional 30 min, diluted with another 20 mL
DCM, washed with 30 mL saturated aqueous NaHCO.sub.3 solution. The
organic layer was separated. The aqueous phase was further
extracted with DCM (3.times.20 mL). The combined organic phase was
washed with brine and dried with anhydrous Na.sub.2SO.sub.4. After
removing volatile components and without any purification, the
crude methanesulfonate was redissolved with 25 mL acetone. To the
resultant reaction mixture was added sodium bicarbonate (160 mg,
1.95 mmol), sodium sulfite (147 mg, 1.17 mmol) and sodium iodide
(580 mg, 3.9 mmol). The suspension was heated to 50.degree. C. and
stirred for about 3 hr under argon. Upon the completion of the
reaction, 20 mL water was added and the resultant mixture was
concentrated under reduced pressure. The residual mixture was then
extracted with ethyl acetate (3.times.30 mL). The combined organic
phase was washed with brine and dried with anhydrous
Na.sub.2SO.sub.4. Removal of the volatile components, followed by
purification with silica gel chromatography (hexane:EtOAc=4:1 then
3:1) yield the final product 6.
[0060] 6 b R=Et, 70% yield. [.alpha.].sub.D.sup.17.9-10.9 (c 0.91,
CHCl.sub.3); .sup.1H-NMR (600 MHz, CDCl.sub.3, rotamers): .delta.
1.18-1.23 (m, 3H), 1.30 (s, 3H), 1.47 (s, 3H), 1.59-1.63 (m, 1H),
1.92-1.94 (m, 0.4H), 2.00-2.04 (m, 0.6H), 2.17 (br, 0.4H), 2.43
(br, 0.6H), 3.02-3.03 (m, 0.4H), 3.09-3.13 (m, 1H), 3.15-3.17 (m,
0.6H), 3.23-3.30 (m, 3H), 3.42 (s, 2H), 4.12-4.19 (m, 1H), 4.44 (d,
0.4H, J=5.8 Hz), 4.52 (d, 0.6H, J=5.8 Hz), 4.56 (d, 0.4H, J=5.8
Hz), 4.61 (d, 0.6H, J=5.8 Hz), 4.90 (s, 0.4H), 4.96 (s, 0.6H),
5.10-5.14 (m, 1.6H), 5.19 (d, 0.4H, J=12.2 Hz), 7.31-7.38 (m, 5H);
.sup.13C-NMR (150 MHz, CDCl.sub.3, rotamers): .delta. 14.92, 15.47,
25.10, 25.17, 26.63, 26.66, 37.50, 37.78, 38.53, 38.62, 55.78,
55.98, 67.00, 67.47, 83.82, 83.98, 84.59, 84.68, 85.72, 85.76,
110.24, 110.44, 112.45, 112.59, 127.89, 128.13, 128.40, 128.65,
128.72, 136.75, 136.99, 155.72, 156.33. MS (ESI) m/z: 556
[M+Na].sup.+; HRMS: calculated for C.sub.22H.sub.32NO.sub.6NaI
([M+Na].sup.+) 556.1172. found 556.1169.
[0061] Synthesis of 7. n-Butyllithum (500 .mu.L, 1.6 M in hexane)
was added dropwise to a stirred solution of
(2R)-2,5-dihydro-2-isopropyl-3,6-dimethoxypyrazine (150 .mu.L, 0.83
mmol) in 3 mL dry THF at -78.degree. C. under argon atmosphere. The
resultant mixture was allowed to be stirred for additional 5 min.
The obtained yellow solution was subsequently transferred via a
double-tipped needle to stirred slurry of copper (I) cyanide (38
mg, 0.42 mmol) in 2 mL THF at -78.degree. C. under argon. This
mixture was stirred at 0.degree. C. for around 1.5 min to afford
cyanocuprate as a tan colored solution. The reaction was then
immediately cooled down to -78.degree. C. A solution of the iodide
6 (0.28 mmol) in 10 mL dry THF was then added dropwise. The
reaction mixture was stirred at -78.degree. C. for 30 min and then
for 16 h at -25.degree. C. under argon. The reaction was quenched
by adding a 1:9 mixture of aqueous ammonia/saturated aqueous
ammonium chloride (15 mL). The aqueous phase was further extracted
with diethyl ether (3.times.20 mL). The organic layer was combined
and then washed with the 1:9 mixtures of concentrated aqueous
ammonia/saturated aqueous ammonium chloride, followed by brine, and
then dried with anhydrous Na.sub.2SO.sub.4. After removing the
volatile components with rotavapor, the crude product was purified
by silica gel flash chromatography (hexane:EtOAc=4:1 then 3:1)
afforded the desired product 7 as colorless oil.
[0062] 7b R=Et, 79% yield. [.alpha.].sub.D.sup.17.9+6.75 (c 1.01,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 64.degree. C.):
.delta. 0.65 (d, 3H, J=6.8 Hz), 0.99 (d, 3H, J=6.8 Hz), 1.10 (t,
3H, J=7.0 Hz), 1.25 (s, 3H), 1.37 (s, 3H), 1.45-1.48 (m, 1H),
1.50-1.60 (m, 3H), 1.71-1.75 (m, 1H), 1.82-1.85 (m, 1H), 2.15-2.20
(m, 1H), 3.14-3.19 (m, 2H), 3.25 (s, 3H), 3.60 (s, 3H), 3.61 (s,
3H), 3.89 (t, 1H, J=3.6 Hz), 3.98 (dd, 1H, J=10.8 Hz, 4.0 Hz),
3.99-4.01 (m, 2H), 4.53 (d, 1H, J=5.9 Hz), 4.55 (d, 1H, J=5.9 Hz),
4.86 (s, 1H), 5.09 (s, 2H), 7.30-7.31 (m, 1H), 7.33-7.36 (m, 4H);
.sup.13C-NMR (150 MHz, DMSO-d.sub.6 rotamers): .delta. 14.20,
15.17, 16.38, 19.01, 24.64, 26.23, 27.90, 30.73, 31.03, 37.52,
37.88, 52.07, 53.83, 54.03, 54.27, 59.77, 65.81, 66.13, 83.29,
83.41, 83.66, 83.70, 84.90, 109.05, 109.22, 111.32, 127.22, 127.60,
127.68, 128.21, 128.36, 136.97, 137.26, 155.52, 162.76, 163.03,
163.11; MS (ESI) m/z: 590 [M+H].sup.+; HRMS: calculated for
C.sub.31H.sub.48N.sub.3O.sub.8 ([M+H].sup.+) 590.3441. found
590.3440.
[0063] Synthesis of 9. To a solution of the dihydropyrazine 7 (0.25
mmol) in 8 mL acetonitrile was added 6 mL 0.25 M aqueous HCl. This
mixture was stirred for 2 hr at ambient temperature and then
neutralized with 10 mL saturated aqueous NaHCO.sub.3 solution at
0.degree. C. The crude product was extracted with 20 mL ethyl
acetate. The resultant aqueous phase was further extracted with
ethyl acetate (3.times.20 mL). The combined organic layer was
washed with brine, dried with anhydrous Na.sub.2SO.sub.4, and then
concentrated under reduced pressure to give the corresponding crude
.alpha.-amino methyl carboxylate 8. Without further purification,
the .alpha.-amino methyl carboxylate was dissolved in 6 mL THF and
cooled down to 0.degree. C. Saturated aqueous NaHCO.sub.3 solution
of 0.4 mL was then added, followed by addition of 30 .mu.L benzyl
chloroformate. The resultant mixture was allowed to spontaneously
warm up at ambient temperature and stirred for additional 8 hr. To
the reaction mixture were added 20 mL ethyl acetate and 20 mL
water. The organic layer was separated. The aqueous layer was
further extracted with ethyl acetate (3.times.20 mL). The combined
organic layer was washed with brine and dried with anhydrous
Na.sub.2SO.sub.4. After removing volatile solvents, the crude
reaction product was purified by silica gel flash chromatography on
(hexane:EtOAc=3:1 then 3:2) to afford 9.
[0064] 9b R=Et, 78% yield. [.alpha.].sub.D.sup.17.9+11.10 (c 1.21,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6 at 64.degree. C.):
.delta. 1.10 (t, 3H, J=7.0 Hz), 1.25 (s, 3H), 1.38 (s, 3H),
1.54-1.68 (m, 5H), 1.83-1.85 (m, 1H), 3.10-3.15 (m, 1H), 3.16-3.21
(m, 1H), 3.24 (s, 3H), 3.62 (s, 3H), 3.98 (dd, 2H, J=10.8 Hz, 3.8
Hz), 4.09-4.12 (m, 1H), 4.53 (d, 1H, J=6.0 Hz), 4.55 (d, 1H, J=6.0
Hz), 4.85 (s, 1H), 5.04 (s, 2H), 5.10 (d, 2H, J=10.2 Hz), 7.29-7.37
(m, 10H), 7.40 (br, 1H); .sup.13C-NMR (150 MHz, DMSO-d.sub.6
rotamers): .delta.4.11, 14.38, 15.30, 24.63, 26.25, 29.04, 37.21,
37.72, 51.89, 53.38, 54.35, 65.51, 65.91, 66.12, 83.20, 83.27,
83.73, 83.77, 84.83, 109.09, 109.22, 111.32, 111.35, 127.16,
127.26, 127.59, 127.72, 127.80, 127.89, 128.26, 128.38, 128.43,
136.93, 137.05, 137.24, 155.49, 156.18, 172.75, 172.81; MS (ESI)
m/z: 651 [M+Na].sup.+; HRMS: calculated for
C.sub.33H.sub.44N.sub.2O.sub.10Na ([M+Na].sup.+; 651.2894. found
651.2905.
[0065] Synthesis of 10. To a stirred solution of 9 (0.2 mmol) in 20
mL dioxane was added 4 M aqueous hydrochloric acid (5 mL, 20 mmol)
at ambient temperature. The resultant mixture was stirred at
ambient temperature for additional 40 h. The reaction was then
quenched with saturated aqueous NaHCO.sub.3 at 0.degree. C. and was
concentrated under reduced pressure. The crude product was
extracted with ethyl acetate (3.times.40 mL). The combined organic
phase was dried with anhydrous Na.sub.2SO.sub.4. The corresponding
crude triol product was obtained after removing volatile components
under reduced pressure. The crude product was dissolved in 5 mL dry
pyridine and cooled down to 0.degree. C. Acetic anhydride (370
.mu.L, 4 mmol) was then added. The resultant reaction mixture was
stirred at 0.degree. C. at ambient temperature overnight, and then
concentrated under reduced pressure at ambient temperature. After
adding saturated NaHCO.sub.3 (30 mL), the residual mixture was
extracted with ethyl acetate (3.times.40 mL). The combined organic
phase was washed with brine, dried with anhydrous Na.sub.2SO.sub.4.
After removing volatile solvents with ratovapor, the crude product
was purified by silica gel chromatography (hexane:EtOAc=2:1 then
1:1) to yield the triacetate derivative of 10 as a 1'-anomeric
mixture.
[0066] Triacetate 10b R=Et, 62% yield.
[.alpha.].sub.D.sup.17.9+12.88 (c 0.94, CHCl.sub.3); .sup.1H-NMR
(600 MHz, DMSO-d.sub.6, 74.degree. C.): .delta. 1.07 (t, 3H, 7.0
Hz), 1.56-1.62 (m, 3H), 1.64-1.69 (m, 2H), 1.98-2.05 (m, 1H), 2.00
(s, 3H), 2.04 (s, 3H), 2.07 (s, 3H), 3.07-3.11 (m, 1H), 3.16-3.21
(m, 1H), 3.61 (s, 3H), 3.92-3.94 (m, 1J), 4.01-4.05 (m, 1H),
4.05-4.07 (m, 1H), 5.04-5.09 (m, 5H), 5.26 (dd, 1H, J=5.1 Hz, 1.2
Hz), 6.02 (d, 1H, J=1.2 Hz), 7.29-7.32 (m, 1H), 7.33-7.37 (m, 10H);
.sup.13C-NMR (150 MHz, DMSO-d.sub.6 rotamers): .delta.4.11, 15.16,
20.24, 20.27, 20.36, 20.85, 27.75, 27.86, 29.09, 37.24, 37.63,
51.89, 53.57, 65.52, 65.99, 66.07, 73.58, 73.70, 73.95, 78.65,
78.84, 98.11, 127.27, 127.76, 127.79, 128.89, 128.31, 128.38,
128.43, 136.94, 137.01, 137.16, 155.38, 156.17, 168.97, 169.35,
169.62, 172.62, 172.69; MS (ESI) m/z: 723 [M+Na].sup.+; HRMS:
calculated for C.sub.35H.sub.44N.sub.2O.sub.13Na ([M+Na].sup.+)
723.2741. found: 723.2770.
[0067] Synthesis of 12. To an oven-dried flask was added
N.sup.6-benzoyladenine (44 mg, 0.18 mmol), hexamethyldisilazane (3
mL) and then dry pyridine (1 mL). The suspension was heated to
115.degree. C. under argon to give a clear solution, which was
stirred to 115.degree. C. for additional 3 h. After removing
volatile components to dryness, the residual volatile component was
then coevaporated with toluene (3.times.5 mL). The mixture was
subject to high vacuum for another 2 h. The resultant white solid
was added to the solution of the triacetate derivative 10 as
prepared above (0.037 mmol) and then dissolved in dry
1,2-dichloroethane (15 mL). The resultant suspension was treated
with TMSOTf (33 .mu.L, 0.18 mmol) dropwise under argon. The
reaction mixture was heated at 50.degree. C. for 2 h, cooled down
to ambient temperature, and then quenched with saturated aqueous
NaHCO.sub.3 (20 mL). The organic phase was separated, and the
aqueous phase was further extracted with CH.sub.2Cl.sub.2
(3.times.20 mL). The organic phase was combined, washed with brine
and the dried with anhydrous Na.sub.2SO.sub.4. After removing
volatile components with ratovapor, the crude product was purified
by silica gel chromatography (CH.sub.2Cl.sub.2:MeOH=25:1) to give
12.
[0068] 12b R=Et, 81% yield. [.alpha.].sub.D.sup.17.7+9.4 (c 0.86,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 74.degree. C.):
.delta. 1.09 (t, 3H, J=7.1 Hz), 1.54-1.57 (m, 2H), 1.61-1.66 (m,
2H), 1.99-2.03 (m, 1H), 2.02 (s, 3H), 2.10-2.16 (m, 1H), 2.14 (s,
3H), 3.11 (q, 1H, J=7.1 Hz), 3.18 (q, 1H, J=7.1 Hz), 3.59 (s, 3H),
3.92-3.94 (m, 1H), 4.03-4.07 (m, 2H), 5.03-5.10 (m, 4H), 5.45 (t,
1H, J=7.2 Hz), 6.06 (t, 1H, J=5.4 Hz), 6.25 (d, 1H, J=5.4 Hz),
7.27-7.35 (m, 11H), 7.54 (t, 2H, J=7.7 Hz), 7.63 (t, 1H, J=7.4 Hz),
8.05 (d, 2H, J=7.5 Hz), 8.63 (s, 1H), 8.73 (s, 1H), 10.85 (br, 1H);
.sup.13C-NMR (150 MHz, DMSO-d.sub.6 rotamers): .delta. 14.00,
14.23, 15.18, 20.28, 20.43, 23.72, 24.31, 27.79, 28.26, 28.73,
29.06, 29.12, 30.73, 31.32, 35.54, 35.95, 36.23, 51.87, 53.61,
65.50, 65.97, 71.95, 73.25, 73.32, 79.07, 79.25, 85.76, 85.88,
126.07, 127.13, 127.30, 127.56, 127.75, 127.83, 128.19, 128.27,
128.35, 128.44, 128.53, 128.56, 132.58, 133.25, 136.83, 136.95,
137.17, 143.82, 143.97, 150.73, 151.79, 151.89, 155.42, 156.12,
165.68, 169.42, 169.59, 172.60, 172.67; MS (ESI) m/z: 902
[M+Na].sup.+; HRMS: calculated for C.sub.45H.sub.50N.sub.7O.sub.12
([M+H].sup.+) 880.3517. found: 880.3541.
[0069] Synthesis of 100, 101 and 110. To a stirred solution of 12
(0.02 mmol) in methanol (10 mL) was added potassium carbonate (14
mg, 0.1 mmol). The resultant mixture was stirred at ambient
temperature for 8 h, concentrated to dryness and then redissolved
in 10 mL water. To the mixture was added hydrazine monohydrate (5
.mu.L, 0.1 mmol). The reaction was stirred for 8 h at ambient
temperature, neutralized with 1M aqueous HCl and then concentrated
under reduced pressure. This mixture was then dissolved in 6 mL
ethanol:water (5:1). To this solution was added 20 .mu.L acetic
acid and palladium on activated carbon (15 mg, 10 wt %, wet Degussa
type). The subsequent hydrogenation reaction was carried out with
hydrogen balloon for 12 h. The reaction mixture was filtered
through a short pad of Celite that was pre-washed with 20 mL MeOH
and then 20 mL 0.1% TFA/water. The combined filtrates were
concentrated under reduced pressure. The resultant crude product
was purified by preparative reversed-phase HPLC (XBridge.TM. Prep C
18 5 .mu.m OBD.TM. 19.times.150 mm) as the following: the 0-10%
gradient of acetonitrile in aqueous trifluoroacetic acid (0.1%) in
10 min and a flow rate of 10 mL/min; The fractions containing
desired compound was collected. The volatile solvents were removed
by SpeedVac. The resultant solution was lyophilized to give the
desired products 100, 101 and 110.
[0070] 100 R=Et, 56% yield. .sup.1H-NMR (600 MHz, MeOD): .delta.
1.11 (t, 3H, J=7.2 Hz), 1.93-1.97 (m, 2H), 1.99-2.07 (m, 2H),
2.23-2.27 (m, 1H), 2.28-2.32 (m, 1H), 3.05 (q, 2H, J=7.2 Hz),
3.46-3.48 (m, 1H), 3.97 (t, 1H, J=6.0 Hz), 4.19-4.22 (m, 1H), 4.37
(t, 1H, J=6.0 Hz), 4.70 (dd, 1H, J=5.4 Hz, 3.8 Hz), 5.99 (d, 1H,
J=3.8 Hz), 8.30 (s, 2H); .sup.13C-NMR (150 MHz, MeOD): .delta.
11.53, 26.99, 27.71, 33.51, 41.92, 53.75, 56.89, 74.57, 75.17,
80.91, 91.78, 118.09 (q, J=289.2 Hz), 121.12, 142.79, 150.26,
151.64, 156.02, 162.70 (q, J=35.4 Hz), 171.77; MS (ESI) m/z: 410
[M+H].sup.+; HRMS: calculated for C.sub.17H.sub.28N.sub.7O.sub.5
([M+H].sup.+) 410.2152. found 410.2142.
[0071] 101 R=Me, 52% yield. .sup.1H-NMR (600 MHz, MeOD):
.delta.1.96-2.03 (m, 2H), 2.05-2.08 (m, 2H), 2.25-2.29 (m, 2H),
2.64 (s, 3H), 3.43-3.45 (m, 1H), 3.99-4.03 (m, 1H), 4.19-4.22 (m,
1H), 4.36 (t, 1H, J=5.9 Hz), 4.65 (dd, 1H, J=5.4 Hz, 3.7 Hz), 6.01
(d, 1H, J=3.7 Hz), 8.35 (s, 1H), 8.36 (s, 1H); .sup.13C-NMR (150
MHz, MeOD): .delta. 26.54, 27.60, 31.43, 33.55, 53.59, 58.23,
74.86, 75.01, 80.92, 91.85, 117.99 (q, J=289.7 Hz), 121.15, 143.44,
149.45, 150.10, 154.64, 162.57 (q, J=35.5 Hz), 171.52; MS (ESI)
m/z: 396 [M+H].sup.+; HRMS: calculated for
C.sub.17H.sub.28N.sub.7O.sub.5 ([M+H].sup.+) 396.1995. found:
396.1982.
[0072] 110 R=Bn, 30% yield. .sup.1H-NMR (600 MHz, MeOD): .delta.
1.97-2.10 (m, 4H), 2.31 (ddd, 1H, J=15.8 Hz, 5.8 Hz, 3.2 Hz),
2.40-2.45 (m, 1H), 3.57-3.59 (m, 1H), 3.99 (t, 1H, J=6.0 Hz), 4.12
(d, 1H, J=13.0 Hz), 4.20 (d, 1H, J=13.0 Hz), 4.41 (t, 1H, J=6.0
Hz), 4.70 (dd, 1H, J=5.8 Hz, 4.0 Hz), 5.49 (s, 2H), 5.99 (d, 1H,
J=3.8 Hz), 7.10 (d, 2H, J=7.2 Hz), 7.23 (t, 2H, J=7.2 Hz), 7.31 (t,
1H, J=7.2 Hz), 8.20 (s, 1H), 8.33 (s, 1H); .sup.13C-NMR (150 MHz,
MeOD): .delta. 27.09, 27.87, 32.45, 53.71, 54.96, 57.08, 74.33,
74.84, 80.98, 91.88, 121.22, 130.22, 130.55, 130.68, 132.26,
142.88, 150.14, 151.49, 155.86, 162.55 (q, J=35.4 Hz), 171.75; MS
(ESI) m/z: 472 [M+H].sup.+; HRMS: calculated for
C.sub.22H.sub.30N.sub.7O.sub.5 ([M+H].sup.+) 472.2308. found
472.2299.
[0073] Synthesis of 13. A stream of ozone was bubbled through a
stirred solution of the alkene 4 (0.64 mmol) in a 30 mL mixture of
1:1 methanol and CH.sub.2Cl.sub.2 at -78.degree. C. until blue
color persisted over 5 min. Then the solution was flushed with
argon until it became clear, dimethyl sulfide (1 mL) was added into
the solution at -78.degree. C. After removing the dry ice bath, the
reaction mixture was allowed to warm up spontaneously at ambient
temperature and was then stirred overnight. Evaporation of the
volatile chemicals under reduced pressure gave the crude acetal,
which was purified by flash silica gel chromatography
(hexane:EtOAc=2:1 then 1:1) to yield Compound 13 as colorless oil
(215 mg, 77% yield).
[0074] 13 [.alpha.].sub.D.sup.16.6-7.2 (c 1.14, CHCl.sub.3);
.sup.1H-NMR (500 MHz, CDCl.sub.3): .delta.1.31 (s, 3H), 1.48 (s,
3H), 1.72-7.74 (m, 1H), 1.76-1.81 (m, 1H), 1.84-1.86 (m, 2H), 3.30
(s, 3H), 3.33 (s, 3H), 3.36 (s, 3H), 3.98-4.02 (m, 1H), 4.32 (dd,
1H, J=10.6 Hz, 3.6 Hz), 4.47 (t, 1H, J=5.5 Hz), 4.55 (d, 1H, J=5.9
Hz), 4.60 d, 1H, J=5.9 Hz), 4.97 (s, 1H), 5.10 (d, 1H, J=1.4 Hz),
5.18 (d, 1H, J=9.2 Hz), 7.31-7.36 (m, 5H); .sup.13C-NMR (125 MHz,
CDCl.sub.3): 624.97, 26.47, 37.78, 39.82, 45.90, 52.65, 53.46,
55.32, 66.52, 83.87, 84.57, 85.44, 102.45, 110.09, 112.35, 128.02,
128.08, 128.46, 136.69, 155.82; MS (ESI) m/z: 462 [M+Na].sup.+;
HRMS: calculated for C.sub.22H.sub.33NO.sub.8Na ([M+Na].sup.+)
462.2104. found 462.2089.
[0075] Compound 14 was synthesized through the procedure for
intermediate 4 using allyl iodide.
[0076] 56% yield. [.alpha.].sub.D.sup.17.5+7.8 (c 1.93,
CHCl.sub.3); .sup.1H-NMR (500 MHz, CDCl.sub.3): .delta.1.31 (s,
3H), 1.48 (s, 3H), 1.72-7.74 (m, 1H), 1.76-1.81 (m, 1H), 1.84-1.86
(m, 2H), 3.30 (s, 3H), 3.33 (s, 3H), 3.36 (s, 3H), 3.98-4.02 (m,
1H), 4.32 (dd, 1H, J=10.6 Hz, 3.6 Hz), 4.47 (t, 1H, J=5.5 Hz), 4.55
(d, 1H, J=5.9 Hz), 4.60 d, 1H, J=5.9 Hz), 4.97 (s, 1H), 5.10 (d,
1H, J=1.4 Hz), 5.18 (d, 1H, J=9.2 Hz), 7.31-7.36 (m, 5H);
.sup.13C-NMR (125 MHz, CDCl.sub.3): 624.97, 26.47, 37.78, 39.82,
45.90, 52.65, 53.46, 55.32, 66.52, 83.87, 84.57, 85.44, 102.45,
110.09, 112.35, 128.02, 128.08, 128.46, 136.69, 155.82; MS (ESI)
m/z: 502 [M+Na].sup.+; HRMS: calculated for
C.sub.25H.sub.37NO.sub.8Na ([M+Na].sup.+) 502.2417. found
502.2404.
[0077] Synthesis of 15. A solution of the acetal 14 (170 mg, 0.36
mmol) and iodine (9 mg, 0.035 mmol) in 15 mL acetone (ACS reagent,
.ltoreq.0.5% H.sub.2O) was stirred at room temperature for 20 min.
The reaction process was carefully monitored by
TLC(CH.sub.2Cl.sub.2/MeOH=15:1). When most of the starting material
(.about.90%) was consumed, the reaction was quenched with 5%
aqueous Na.sub.2S.sub.2O.sub.3 (5 mL). The reaction mixture was
concentrated under reduced pressure and then diluted with 50 mL
ethyl acetate. The mixture was washed with 20 mL H.sub.2O and then
20 mL brine. The resultant organic layer was dried with anhydrous
Na.sub.2SO.sub.4. The solvent was removed to give the crude
aldehyde. Without further purification, the aldehyde product was
reduced with NaBH.sub.4 as described for 5. After flash
chromatography workup (hexane:EtOAc=1:1 then 1:2), 15 was obtained
in 79% yield (12 mg acetal 7 was recovered).
[0078] 15 [.alpha.].sub.D.sup.17.5+14.7 (c 1.08, CHCl.sub.3);
.sup.1H-NMR (600 MHz, DMSO-d.sub.6 at 74.degree. C.): 1.25 (s, 3H),
1.38 (s, 3H), 1.60-1.65 (m, 2H), 1.80-1.85 (m, 1H), 1.88-1.92 (m,
1H), 3.27 (s, 3H), 3.38 (q, 2H, J=6.0 Hz), 3.77 (dt, 1H, J=6.2 Hz,
1.2 Hz), 3.79 (dt, 1H, J=6.2 Hz, 1.2 Hz), 4.01 (dd, 1H, J=10.4 Hz,
4.2 Hz), 4.05 (br, 1H), 4.08 (t, 1H, J=4.8 Hz), 4.51 (d, 1H, J=6.0
Hz), 4.54 (d, 1H, J=6.0 Hz), 4.85 (s, 1H), 5.08 (dd, 1H, J=10.2 Hz,
1.4 Hz), 5.10 (s, 2H), 5.17 (dd, 1H, J=17.2 Hz, 1.2 Hz), 5.83-5.89
(m, 1H), 7.30-7.31 (m, 1H), 7.34-7.37 (m, 4H); .sup.13C-NMR (150
MHz, DMSO-d.sub.6 rotamers): .delta. 25.08, 26.60, 36.09, 37.81,
55.56, 58.90, 67.64, 76.97, 77.23, 77.48, 83.75, 84.55, 85.68,
110.16, 112.44, 117.54, 128.00, 128.20, 128.66, 135.18, 136.62,
156.54, 157.39; MS (ESI) m/z: 458 [M+Na].sup.+; HRMS: calculated
for C.sub.23H.sub.33NO.sub.7Na ([M+Na].sup.+) 458.2155. found
458.2156.
[0079] Compound 16 was obtained from 15 by a series of steps
analogous to steps 5.fwdarw.12 of Scheme 1.
[0080] 16 98% yield. [.alpha.].sub.D.sup.17.7+4.90 (c 1.15,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 64.degree. C.):
.delta. 1.52-1.54 (m, 2H), 1.62-1.65 (m, 2H), 1.99-2.06 (m, 1H),
2.03 (s, 3H), 2.10-2.16 (m, 1H), 2.11 (s, 3H), 3.59 (s, 3H), 3.69
(dd, 1H, J=15.8 Hz, 6.4 Hz), 3.81 (dd, 1H, J=15.8 Hz, 6.4 Hz),
4.03-4.09 (m, 3H), 5.03-5.09 (m, 5H), 5.14 (d, 1H, J=17.2 Hz), 5.44
(t, 1H, J=5.2 Hz), 5.80-5.87 (m, 1H), 6.07 (t, 1H, J=5.6 Hz), 6.25
(d, 1H, J=5.5 Hz), 7.28-7.35 (m, 10H), 7.42 (br, 1H), 7.55 (t, 2H,
J=7.9 Hz), 7.64 (t, 1H, J=7.4 Hz), 8.05 (d, 2H, J=7.4 Hz), 8.64 (s,
1H), 8.74 (s, 1H), 10.93 (s, 1H); .sup.13C-NMR (150 MHz,
DMSO-d.sub.6 rotamers): .delta. 20.23, 20.37, 27.71, 28.98, 30.68,
35.53, 51.82, 53.55, 65.48, 66.19, 66.26, 71.97, 73.09, 73.20,
79.15, 85.71, 85.83, 116.89, 126.02, 127.09, 127.29, 127.71,
127.80, 128.16, 128.31, 128.48, 128.51, 132.52, 133.25, 136.70,
136.92, 143.73, 143.89, 150.70, 151.77, 151.85, 155.63, 156.08,
165.64, 169.35, 169.47, 172.61; MS (ESI) 914 [M+Na].sup.+; HRMS:
calculated for C.sub.46H.sub.50N.sub.7O.sub.12 ([M+H].sup.+)
892.3517. found: 892.3495.
[0081] Compound 102 was obtained from 16 by the procedure described
for conversion of 12 to 100 above and in Scheme 1.
[0082] 102 .sup.1H-NMR (500 MHz, MeOD): .delta. 0.83 (t, 3H, J=7.4
Hz), 1.42-1.49 (m, 1H), 1.52-1.59 (m, 1H), 1.94-2.09 (m, 4H),
2.21-2.26 (m, 1H), 2.29-2.35 (m, 1H), 2.92 (t, 2H, J=8.0 Hz)
3.44-3.48 (m, 1H), 4.01 (t, 1H, J=6.0 Hz), 4.19-4.22 (m, 1H), 4.40
(t, 1H, J=6.0 Hz), 4.67 (dd, 1H, J=5.4 Hz, 3.4 Hz), 6.02 (d, 1H,
J=3.4 Hz), 8.35 (s, 1H), 8.36 (s, 1H); .sup.13C-NMR (150 MHz,
MeOD): .delta. 11.20, 20.77, 27.05, 27.64, 33.32, 48.23, 53.54,
57.29, 74.83, 75.16, 80.91, 91.90, 117.92 (q, J=289.4 Hz), 121.11,
143.47, 149.35, 150.09, 154.55, 162.44 (q, J=35.8 Hz), 171.50; MS
(ESI) m/z: 424 [M+H].sup.+; HRMS: calculated for
C.sub.18H.sub.30N.sub.7O.sub.5 ([M+H].sup.+) 424.2308. found
424.2296.
[0083] The synthesis of 23 (as shown in Scheme 6). To a solution of
oxazolidinone 1 (2.0 g, 4.5 mmol) in dry diethyl ether (60 mL) was
added anhydrous ethanol (316 .mu.L, 5.4 mmol) and LiBH.sub.4 (2.0M
in THF, 2.7 mL, 5.4 mmol) at 0.degree. C. The reaction was stirred
for 30 min and allowed to warm to room temperature and stirred for
additional 3 h under argon. The reaction was quenched slowly with
aqueous sodium hydroxide (1.0M, 40 mL) and allowed to stir until
both layers were clear. The aqueous phase was separated and
extracted with ethyl acetate (40 mL.times.3). All the organic
layers were combined, washed with brine and dried over MgSO.sub.4.
After the removal of organic solvent, the residue was purified by
flash silica gel chromatography (hexane:EtOAc=2:1) to give an
intermediate alcohol as a colorless oil (1.0 g, 3.68 mmol, 82%
yield).
[0084] .sup.1H-NMR (500 MHz, CDCl.sub.3): .delta. 1.30 (s, 3H),
1.47 (s, 3H), 1.53-1.58 (m, 1H), 1.61-1.67 (m, 1H), 1.82-1.87 (m,
2H), 2.08-2.14 (m, 1H), 2.16-2.22 (m, 1H), 3.35 (s, 3H), 3.62 (dd,
2H, J=7.9 Hz, 5.0 Hz), 4.32 (dd, 1H, J=10.0 Hz, 5.3 Hz), 4.52 (d,
1H, J=5.9 Hz), 4.60 (d, 1H, J=5.9 Hz), 4.93 (s, 1H), 5.01-5.08 (m,
2H), 5.74-5.81 (m, 1H); .sup.13C-NMR (125 MHz, CDCl.sub.3): .delta.
24.98, 26.52, 35.90, 36.02, 37.38, 55.32, 64.42, 76.78, 77.03,
77.28, 84.69, 84.88, 85.45, 109.95, 112.29, 116.63, 136.55; MS
(ESI) m/z: 295 ([M+Na]+; HRMS: calculated for C14H24NO5Na ([M+Na]+)
295.1521. found 295.1529.
[0085] To the solution of alcohol (600 mg, 2.2 mmol) in
dichloromethane (30 mL) was added NaHCO.sub.3 (1.8 g, 22 mmol) and
Dess-Martin periodinane
(1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one) (1.12 g,
2.64 mmol) at 0.degree. C. The suspension was stirred for 40 min at
room temperature under nitrogen. The solution of
Na.sub.2S.sub.2O.sub.3 (2.0M, 2 mL) and saturated aqueous
NaHCO.sub.3 solution (10 mL) was added to above suspension and
stirred for 15 min. The system was diluted with water (20 mL) and
separated. The aqueous phase was extracted with dichloromethane (30
mL.times.3). All the organic layers were combined and washed with
brine and dried over Na2SO4. After the concentration on rotavapor
at room temperature, the crude aldehyde 22 was used to next run
directly (Note: the concentrated aldehyde may decompose over time
at room temperature).
[0086] The aldehyde 22 was dissolved in dry 1,2-dichloroethane (20
mL) and benzylamine (252 .mu.L, 2.3 mmol), and sodium
triacetoxyborohydride (653 mg, 3.1 mmol) were added in turn. The
suspension was stirred at room temperature under a nitrogen
atmosphere for 2 hours. TLC analysis showed the reaction had
completed. The reaction mixture was quenched by adding aqueous
saturated NaHCO.sub.3 (20 mL). After the separation, the aqueous
phase was extracted with dichloromethane (30 mL.times.3). The
combined organic solvent was washed with brine and dried over
MgSO.sub.4. The crude compound was obtained after concentration,
redissoved in THF (25 mL) and cooled down to 0.degree. C. Saturated
NaHCO.sub.3 (5 mL) was added to above solution followed by benzyl
chloroformate (400 .mu.L, 3.3 mmol). The suspension was allowed to
warm to room temperature spontaneously and stirred for 12 h. The
mixture was diluted with water (30 mL) and extracted with ethyl
acetate (40 mL.times.3). The combined organic layers were washed
with brine, then dried over anhydrous Na.sub.2SO.sub.4 and
evaporated. The residue was purified by silica gel chromatography
(hexane:EtOAc=4:1.about.2:1) to furnish compound 23 as colorless
oil (671 mg, 61% over 3 steps)
[0087] Compound 23 [.alpha.].sub.D.sup.18.6-13.6 (c 0.98,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 74.degree. C.):
.delta. 1.26 (s, 3H), 1.37 (s, 3H), 1.38-1.42 (m, 1H), 1.48-1.52
(m, 1H), 3.20-3.24 (m, 2H), 3.22 (s, 3H), 4.08 (t, 1H, J=7.5 Hz),
4.38 (d, 1H, J=5.8 Hz), 4.48 (d, 2H, J=2.3 Hz), 4.50 (d, 1H, J=5.8
Hz), 4.84 (s, 1H), 4.96-4.99 (m, 2H), 5.13 (s, 1H), 5.67-5.72 (m,
2H), 7.23-7.27 (m, 3H), 7.30-7.34 (m, 7H); .sup.13C-NMR (150 MHz,
DMSO-d6 rotamers): .delta. 24.72, 26.30, 32.78, 33.02, 36.25,
49.74, 50.02, 50.21, 50.42, 54.47, 65.39, 66.38, 66.55, 83.64,
83.94, 84.04, 84.77, 84.83, 108.95, 111.31, 11672, 116.86, 127.01,
127.10, 127.16, 127.48, 127.68, 127.78, 127.83, 128.29, 128.37,
128.48, 128.51, 136.10, 136.26, 136.86, 138.15, 155.90, 156.11; MS
(ESI) m/z: 518 ([M+Na].sup.+); HRMS: calculated for C29H37NO6Na
([M+Na].sup.+) 518.2519. found 518.2522.
[0088] Compounds 201 and 202 were synthesized from intermediate 23,
via intermediate 24, as shown in Scheme 6.
[0089] Compound 201 .sup.1H-NMR (500 MHz, D.sub.2O): .delta.
1.49-1.55 (m, 2H), 1.84-1.97 (m, 5H), 2.96 (dd, 1H, J=12.8 Hz, 6.9
Hz), 3.05 (dd, 1H, J=12.8 Hz, 5.4 Hz), 3.82 (t, 1H, J=6.0 Hz),
3.99-4.03 (m, 1H), 4.19-4.22 (m, 1H), 4.36 (t, 1H, J=5.9 Hz), 4.65
(dd, 1H, J=5.4 Hz, 3.7 Hz), 6.01 (d, 1H, J=3.7 Hz), 8.35 (s, 1H),
8.36 (s, 1H); .sup.13C-NMR (150 MHz, D.sub.2O): .delta. 25.84,
26.88, 33.12, 33.65, 41.94, 53.79, 73.35, 81.18, 88.81, 115.28,
116.25 (q, J=289.8 Hz), 119.02, 142.76, 144.67, 148.17, 150.08,
162.97 (q, J=35.2 Hz), 173.33; MS (ESI) m/z: 396 [M+H].sup.+; HRMS:
calculated for C.sub.16H.sub.25N.sub.7O.sub.5 ([M+H].sup.+)
396.1995. found: 396.1982.
[0090] Compound 202 .sup.1H-NMR (600 MHz, MeOD): .delta. 1.46-1.56
(m, 2H), 1.76-1.80 (m, 1H), 1.83-1.91 (m, 2H), 1.92-2.03 (m, 2H),
2.94-3.00 (m, 1H), 3.82 (t, 1H, J=7.0 Hz), 3.95 (d, 1H, J=13.0 Hz),
3.98-4.00 (m, 1H), 4.04 (d, 1H, J=13.0 Hz), 4.08 (t, 1H, J=5.7 Hz),
4.49 (dd, 1H, J=5.4 Hz, 4.0 Hz), 5.84 (d, 1H, J=4.0 Hz), 7.12 (t,
2H, J=7.4 Hz), 7.20 (t, 2H, J=7.4 Hz), 7.25 (d, 1H, J=7.4 Hz), 8.09
(s, 1H), 8.16 (s, 1H); .sup.13C-NMR (150 MHz, MeOD): .delta. 27.90,
28.72, 34.32, 34.69, 52.07, 53.01, 54.07, 74.88, 75.36, 82.07,
91.65, 121.19, 130.37, 130.83, 130.90, 132.10, 142.68, 150.18,
151.31, 162.40 (q, J=35.4 Hz), 171.94; MS (ESI) m/z: 486
[M+H].sup.+; HRMS: calculated for C.sub.23H.sub.32N.sub.7O.sub.5
([M+H].sup.+) 486.2465. found: 486.2464.
[0091] The synthesis of 204.
[0092] Compound 31 (Scheme 8) was derived from intermediate 40 in a
similar way to the preparation of compound 27 in scheme 7.
[0093] Compound 31 [.alpha.].sub.D.sup.18.3-5.2 (c 0.93,
CHCl.sub.3); .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 84.degree. C.):
.delta. 1.31-1.39 (m, 2H), 1.64-1.67 (m, 1H), 1.75-1.80 (m, 1H),
1.81-1.88 (m, 2H), 1.92-1.95 (m, 1H), 2.04 (s, 3H), 2.10 (s, 3H),
3.13-3.16 (m, 1H), 3.23-3.26 (m, 1H), 3.66 (s, 3H), 4.15-4.18 (m,
1H), 4.24-4.28 (m, 1H), 4.39 (d, 1H, J=15.6 Hz), 4.45 (d, 1H,
J=15.6 Hz), 5.10 (d, 2H, J=5.5 Hz), 5.41 (t, 1H, J=5.6 Hz), 6.04
(t, 1H, J=5.6 Hz), 6.23 (d, 1H, J=5.6 Hz), 7.12-7.13 (m, 2H),
7.22-7.31 (m, 8H), 7.54 (t, 2H, J=7.6 Hz), 7.63 (t, 1H, J=7.4 Hz),
8.04 (d, 2H, J=7.6 Hz), 8.59 (s, 1H), 8.71 (s, 1H), 9.39 (d, 1H,
J=6.4 Hz), 10.76 (s, 1H); .sup.13C-NMR (150 MHz, DMSO-d.sub.6
rotamers): .delta. 20.25, 20.38, 20.42, 26.70, 27.48, 30.72, 32.41,
32.81, 34.25, 52.29, 52.82, 54.94, 66.38, 66.52, 71.89, 73.17,
79.17, 85.75, 85.85, 115.77 (q, J=286.1 Hz), 126.01, 126.90,
127.12, 127.28, 127.39, 127.51, 127.79, 128.29, 128.32, 128.43,
128.52, 128.54, 132.57, 133.23, 136.77, 137.85, 143.88, 150.71,
151.80, 151.85, 155.87, 156.59 (q, J=36.4 Hz), 165.67, 169.40,
169.51, 169.60, 170.92; MS (ESI) m/z: 940 ([M+Na].sup.+; HRMS:
calculated for C.sub.45H.sub.47N.sub.7O.sub.11F.sub.3
([M+Na].sup.+) 918.3286. found 918.3311.
[0094] A suspension of 20% palladium hydroxide on activated carbon
(25 mg) in a solution of compound 31 (20 mg) in trifluoroethanol
(10 mL) was stirred under hydrogen balloon at room temperature for
16 h. After this period, the mixture was filtered though a pad of
Celite, which was washed with methanol (40 mL). The combined
filtrates were concentrated and redissolved in dichloromethane (5
mL). 1,3-Di-Boc-2-methylisothiourea (8.2 mg, 0.028 mmol) and
triethylamine (8 .mu.L, 0.06 mmol) was added to above solution,
followed by the solution of mercury(II) chloride (7 mg, 0.028 mmol)
in THF (1004). The resulting clear solution was turned to cloudy
after stirring at room temperature for approximately 15 min. The
mixture was attired for additional 2 h and filtered through a short
pad of Celite, and the Celite pad was washed with dichloromethane
(30 mL). The combined filtrates were concentrated under reduced
pressure give a residue which was chromatographed over silica gel
(DCM:MeOH=30:1) to afford compound 33 (12 mg).
[0095] To the solution of compound 33 (12 mg) in methanol (5 mL)
was added 0.2 M lithium hydroxide (1.2 mL). The resulting solution
was stirred at room temperature overnight and then concentrated.
The residue was dissolved in water (3 mL) and hydrazine monohydrate
(3.1 .mu.L) was added. The reaction mixture was stirred for 6 h at
room temperature and water was removed by lyophilization. The
residue was treated with 1.5 mL TFA:H2O (9:1) for 1 h at room
temperature. After this period, the reaction system was diluted
with water (10 mL), then freeze-fried. The residue of 204 was
dissolved in water (2 mL) and purified as described for compound
100.
[0096] Compound 204 .sup.1H-NMR (500 MHz, MeOD): .delta. 1.58-1.62
(m, 2H), 1.88-2.06 (m, 5H), 3.20 (t, 1H, J=1.8 Hz), 3.94 (t, 1H,
J=5.8 Hz), 4.11-4.15 (m, 1H), 4.25 (t, 1H, J=5.7 Hz), 4.75 (dd, 1H,
J=5.4 Hz, 4.0 Hz), 6.00 (d, 1H, J=4.0 Hz), 8.30 (s, 1H), 8.31 (s,
1H); MS (ESI) m/z: 438 [M+H].sup.+; HRMS: calculated for
C.sub.17H.sub.28N.sub.9O.sub.5 ([M+H].sup.+) 438.2213. found:
438.2210.
[0097] Synthesis of 300 as depicted in Scheme 13: To the solution
of alkene (100 mg, 021 mmol) and protected vinyl glycine (100 mg,
0.42 mmol) in dry dichloromethane (20 mL) was added Grubbs 2.sup.nd
catalyst (35 mg, 0.041 mmol) under argon. The resulting dark brown
solution was sealed and heated to reflux for 6 hr and cooled down
to room temperature. The system was concentrated and purified
through silica gel column (hexane:EA=4:1 to 3:1) to yield cross
coupling product 41 (60 mg, 41%). .sup.1H NMR (CDCl.sub.3, 500 MHz,
rotamers): .delta. 1.27 (s, 3H), 1.46 (s, 3H), 1.62-1.64 (m, 1H),
1.78-1.82 (m, 0.4H), 2.00-2.10 (m, 0.6H), 2.14-2.17 (m, 0.4H),
2.20-2.24 (m, 0.6H), 2.28-2.38 (m, 1H), 3.21 (s, 1.3H), 3.30 (s,
1.7H), 3.70 (1.7H), 3.72 (s, 1.3H), 4.08-4.11 (m, 0.4H), 4.12-4.15
(m, 0.6H), 4.25-4.35 (m, 1.4H), 4.41 (d, 0.6H, J=5.0 Hz), 4.47 (d,
0.4H, J=5.0 Hz), 4.54 ((d, 0.6H, J=5.0 Hz), 4.63-4.71 (m, 2H), 4.87
(s, 0.4H), 4.92 (s, 0.6H), 5.10-5.23 (m, 5.5H), 5.37-5.44 (m,
1.5H), 7.26-7.35 (m, 15H); .sup.13C NMR (CDCl.sub.3, 600 MHz,
rotamers): .delta. 14.40, 21.26, 25.02, 25.08, 26.60, 29.90, 52.75,
52.83, 55.37, 55.51, 55.60, 55.70, 60.61, 67.25, 67.76, 83.93,
84.03, 84.26, 84.39, 85.60, 85.65, 110.10, 110.28, 112.39, 112.47,
126.50, 127.62, 128.08, 128.16, 128.36, 128.42, 128.49, 128.66,
128.68, 128.73, 128.77, 131.56, 136.35, 136.64, 136.70, 138.56,
155.61, 155.67, 157.22, 171.38; MS (ESI): 703 ([M+H].sup.+)
[0098] The compound 37 was converted to its fully-protected form as
previously described. HRMS: calculated for
C.sub.51H.sub.52N.sub.7O.sub.12 ([M+H].sup.+) 954.3674. found
954.3672. The deprotection was carried out through hydrogenolysis
and basic treatment to give the final compound 300. .sup.1H-NMR
(600 MHz, MeOD): .delta. 1.65-1.70 (m, 2H), 1.96-2.08 (m, 4H),
2.25-2.29 (m, 2H), 3.43-3.45 (m, 1H), 3.99-4.03 (m, 1H), 4.19-4.22
(m, 1H), 4.36 (t, 1H, J=5.9 Hz), 4.65 (dd, 1H, J=5.4 Hz, 3.7 Hz),
6.00 (d, 1H, J=3.7 Hz), 8.35 (s, 1H), 8.36 (s, 1H); MS (ESI): 396
([M+H].sup.+); HRMS: calculated for C.sub.16H.sub.26N.sub.7O.sub.5
([M+H].sup.+) 396.1995. found 396.1982.
[0099] The compounds described above were tested as described
below:
[0100] Methylation Reaction. The 20 .mu.L methylation reaction was
carried out at ambient temperature using two mixtures: A. 10 .mu.l
of enzyme mixture in the assay buffer containing 50 mM Hepes
(pH=8.0), 0.005% Tween-20, 5 .mu.m/ml BSA and 1 mM TCEP; B. 10
.mu.l of a mixture of 1.5 .mu.M, 0.15 .mu.Ci [.sup.3H-Me]-SAM
cofactor and 3 .mu.M of the corresponding peptide substrate in the
same assay buffer. After A and B were mixed for a designated time
period, the reaction mixture was examined with our filter-paper
assay.
[0101] Conditions for the enzymes:
TABLE-US-00001 [Enzyme mixture] [Enzyme].sub.final Peptide Reaction
Time Enzyme (nM) (nM) Substrate (h) G9a (913-1913) 40 20 H3 (1-21
aa) 1 GLP1 (951-1235) 20 10 H3 (1-21 aa) 1 SUV39H2 (112-410) 10 5
H3 (1-21 aa) 4 SET7/9 Full-length 300 150 H3 (1-21 aa) 3 PRMT1
(10-352) 200 100 RGG 1.5 PRMT3 (211-531) 200 100 RGG 3 CARM1
(19-608) 600 300 H3 (1-40 aa) 7 SET8 (191-352) 2000 1000 H4 (10-30
aa) 8 SETD2 (1347-1711) 500 250 H3 (20-50 aa) 4 SMYD2 Full-length
100 50 p53 (360-393 10 aa)
TABLE-US-00002 H3 (1-21-aa): (SEQ ID NO: 1) ARTKQTARKSTGGKAPRKQLA
RGG: (SEQ ID NO: 2) GGRGGFGGRGGFGGRGGFG H3 (1-40 aa): (SEQ ID NO:
3) ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHR H4 (10-30 aa): (SEQ ID
NO: 4) LGKGGAKRHRKVLRDNIQGIT H3 (20-50 aa): (SEQ ID NO: 5)
ATKAARKSAPATGGVKKPHRYRPGTVALRE p53 (360-393 aa): (SEQ ID NO: 6)
GGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD
[0102] Filter-paper Assay. This assay relies on Whatman P-81 filter
paper, which binds peptides but not SAM. Protein Methyl
Transferases (PMTS) transfer .sup.3H-Me of [.sup.3H-Me]-SAM to
peptide substrates and the resultant .sup.3H-methylated,
filter-paper-bound peptide is quantified with a scintillation
counter. Briefly, 6 .mu.l of the methylation reaction was spotted
onto Whatman P-81 phosphocellulose filter paper (1.2.times.1.2
cm.sup.2) to immobilize the .sup.3H-labeled peptide. After drying
in air for 20 min, the filter paper was immersed into 20 mL of 50
mM Na.sub.2CO.sub.3/NaHCO.sub.3 buffer (pH=9.2), and washed 5 times
for 10 min each time. The washed filter paper was then transferred
to a 20 ml scintillation vial containing 1 mL of distilled water
and 10 mL of Ultima Gold scintillation cocktail or 7 mL
scintillation vial containing 0.5 mL od distilled water and 5 mL of
scintillation cocktail (PerkinElmer). The radioactivity was
quantified by a Beckman LS6000IC liquid scintillation counter.
[0103] Dose-response Curves and IC.sub.50. Twice the PMT
concentration was incubated for 10 min with varied concentration of
inhibitors (0.1-400 .mu.M stocks), into which 10 .mu.l of the PMT
peptide substrate and radioactive cofactor (3 .mu.M of the
corresponding peptide and 1.5 .mu.M, 0.15 .mu.Ci [.sup.3H-Me]-SAM)
were added. After incubating the reaction mixture for the
respective reaction time, the conversion was quantified with the
filter paper assay as described above. The inhibition was expressed
as the percentage between the high control (no inhibition) and the
low control (no enzyme) as follows: Percentage Inhibition=[(high
control-reading)/(high control-low control)].times.100%. Each
experiment was performed in triplicate. The IC.sub.50 values were
obtained by fitting inhibition percentage versus inhibitor
concentration using GraphPad Prism5 software.
[0104] Cellular Assay: HEK-293T cells were grown in DMEM plus 10%
FBS and maintained in a humidified incubator set to 37.degree. C.,
5% CO.sub.2. For assessment of the inhibitor effect, cells were
plated in 6-well plate at a density of 0.5.times.10.sup.6
cells/well in 2 mL of media. The following day the media was
removed and replenish with 2 mL of increasing concentrations of
PropylSinefungin up to 100 .mu.M. Cell were harvested after 24 h
and proceed to do the histone extraction (see below). 8 .mu.g of
the histones were separated on 18% Tris-HCl gels (BIO-RAD),
transferred to 0.2 .mu.M PVDF membranes and blotted with histone 3
lysine 36 tri-methyl antibody (Abcam) or H3 (Millipore) as a
loading control.
[0105] Histone Extraction: The nuclear pellet and cytoplasm extract
were obtained using the Cell Lytic.TM. NuCLEAR Extraction Kit
(SIGMA). Then 40 .mu.L of cold 0.2 M Sulfuric Acid were added and
incubate overnight at 4.degree. C. Then, the samples were
centrifuge at 11000.times.g for 1 min and the supernatant
containing the histones was collected. The concentration was
measured using Quick Start.TM. Bradford 1.times. Dye Reagent
(BIO-RAD).
[0106] The results are shown in the following table, in which
S-adenosyl homocysteine (SAH) and sinefungin (SIN) are
controls:
TABLE-US-00003 Compd SUV39 SMYD2- No. G9a GLP1 SET7/9 SET8 SETD2
PRMT1 PRMT3 H2 CARM1 FL SAH 6.7 0.7 >100 >100 3.0 8.6 39.5
0.6 1.9 ~50 SIN 18.9 32.0 1.1 >100 28.4 1.0 28.2 4.6 0.5 0.2 100
>500 >500 1.7 >100 8.2 55 37.9 95.7 1.4 0.2 101 164.6
373.7 1.4 >100 125.2 83.9 76.2 43.2 1.7 0.5 102 >100 >100
2.2 >100 0.8 9.5 ~100 9.8 3.0 0.5 103 >100 >100 12.6
>100 2.9 >50 ~70 ~100 9.9 0.3 104 32 >100 14.8 >100
11.3 29.9 0.9 16 1.1 3.6 105 >100 >100 16.6 >100 5.2 1.9
1.9 13.3 0.09 TBD 106 >50 37.4 33.3 >100 .ltoreq.1.5 3.7 TBD
37.5 0.06 TBD 107 >100 >100 >100 >100 >100 >100
>100 >100 ~50 ~50 109 >100 >100 0.19 >100 37 2 1.8
33 0.05 1.7 110 >100 >100 >100 >100 0.5 >100 5.1
>100 22.4 4 111 >100 >100 >100 >100 46.5 >100 33
>100 ~80 >100 116a >100 >100 0.7 >100 >100
>100 >100 >100 >100 2.5 116b >100 >100 0.2
>100 >100 >100 >100 >100 >100 3 116c >100
>100 0.4 >100 >100 >100 >100 >100 >100 3 116d
>100 >100 1.1 >100 >100 >100 >100 >100 61.4
<0.5 116e >100 >100 10.3 >100 ~100 >100 9.7 >100
42 9 201 >100 >100 27.6 >100 136.1 2.5 13.1 24.5 0.1
<0.2 202 >100 >100 >100 >100 10.8 15.25 3.4 10.1
0.09 1.6 203 ~25 ~12.5 9.6 21.8 4.2 >100 7.6 10.7 5.2 1 204 32
24.2 34.8 >100 24.4 1.9 0.7 8.6 0.02 0.2 205 >100 >100
>100 >100 67.9 ~50 9.4 TBD 32.7 ~12.5 300 ~100 ~100 ~70 ~70
21.2 18.75 9.9 35.13 4.4 37
[0107] Compounds that show selective inhibition of one or a few
families of PMTs are of greater interest as candidates for use in
therapy, since it is believed that broad spectrum inhibition is
likely to be associated with a higher probability of side effects.
In this regard, compounds described above as 201, 202, 204, 109,
105 and 106 are of interest because of their apparent
selectivity--among the subset of PMTs screened--for CARM1
inhibition. Analogously, the deazapurines (116a, 116b and 116c)
appear to be selective for SET7/9.
[0108] Compound 102, which, along with 110, is selective for SETD2,
was tested in vivo and showed activity in inhibiting histone
methyltransferase.
Sequence CWU 1
1
6121PRTArtificial SequenceSynthetic peptide 1Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln
Leu Ala 20 219PRTArtificial SequenceSynthetic peptide 2Gly Gly Arg
Gly Gly Phe Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly 1 5 10 15 Gly
Phe Gly 340PRTArtificial SequenceSynthetic peptide 3Ala Arg Thr Lys
Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys
Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr 20 25 30
Gly Gly Val Lys Lys Pro His Arg 35 40 421PRTArtificial
SequenceSynthetic peptide 4Leu Gly Lys Gly Gly Ala Lys Arg His Arg
Lys Val Leu Arg Asp Asn 1 5 10 15 Ile Gln Gly Ile Thr 20
530PRTArtificial SequenceSynthetic peptide 5Ala Thr Lys Ala Ala Arg
Lys Ser Ala Pro Ala Thr Gly Gly Val Lys 1 5 10 15 Lys Pro His Arg
Tyr Arg Pro Gly Thr Val Ala Leu Arg Glu 20 25 30 634PRTArtificial
SequenceSynthetic peptide 6Gly Gly Ser Arg Ala His Ser Ser His Leu
Lys Ser Lys Lys Gly Gln 1 5 10 15 Ser Thr Ser Arg His Lys Lys Leu
Met Phe Lys Thr Glu Gly Pro Asp 20 25 30 Ser Asp
* * * * *